The present invention relates to prodrugs of anti-proliferative agents comprising a phospholipid moiety covalently linked, via a bridging group, to an anti-proliferative agent, such that the active species is preferentially released, preferably by enzymatic cleavage, at the required site of action.
The invention further relates to pharmaceutical compositions comprising said prodrugs and to the use thereof for the treatment of diseases and disorders related to inflammatory, degenerative or atrophic conditions and to uncontrolled cell growth.
Anti-proliferative drugs, also known as anti-metabolites, act by inhibiting crucial metabolic processes and are commonly used in the treatment of diseases involving abnormal cell proliferation, such as tumors. Many anti-proliferative drugs are also useful as anti-inflammatory agents, exerting their effect by suppressing local or even systemic inflammatory responses mediated by the immune system However, the utility of these drugs is severely hampered by their excessive toxicity and adverse side effects on healthy cells of the treated patient.
Methotrexate (MTX) is an effective anti-proliferative drug commonly used in cancer therapy. MTX is also known as a leading anti-inflammatory drug, and is currently the main drug used in the treatment of rheumatoid arthritis (RA). It is the drug of first choice for treating children with recalcitrant juvenile RA.
Methotrexate is an analogue of dihydrofolate that inhibits the enzyme dihydrofolate reductase (DHFR), thus depleting intracellular tetrahydrofolate (FH4) which is an essential co-factor required for the de novo synthesis of purine nucleotides.
Another anti-metabolite widely used in cancer chemotherapy is the pyrimidine analog fluorodeoxyuridine (FUdR). FUdR is converted in vivo into fluorodeoxyuridylate (F-dUMP) which is an analog of the al substrate dUMP and irreversibly inhibits thymidylate synthase.
MTX and FUdR are valuable drugs in the treatment of many rapidly growing tumors, however, their use is limited by the frequency and severity of side effects. Unwanted side effects include toxicity to all rapidly dividing normal cells, such as stem cells in the bone marrow, epithelial cells of the intestinal tract, etc. Folic acid antagonists are also toxic to developing embryos. Treatment with MTX is especially problematic in patients having chronic debilitating inflammatory diseases that require prolonged therapy, such diseases as rheumatoid arthritis, asthma, dermatological diseases such as psoriasis and gastrointestinal inflammation such as Crohn""s disease. These patients may suffer from induced nephrotoxicity, due to precipitation of the drug or its metabolites in the renal tubes, and from hepatic fibrosis and cirrhosis.
Another major problem in chemotherapy, which is particularly relevant in the case of anti-metabolites, is inherent or acquired resistance of tumors to cytotoxic drugs. For example, development of resistance to MTX frequently follows prolonged exposure to this drug. Resistance may be due to new mutations induced by the clinical treatment or to positive selection, by the treatment regimen, of pre-existing resistant mutant cell. Known mechanisms for development of resistance involve impaired transport of MTX into cells, e.g. by mutations in the Reduced Folates Carrier (RFC), over expression of the target enzyme DHFR, or mutations in the enzyme responsible for polyglutamination of reduced folates (FPGS).
A more severe problem in the clinic is the phenomenon of multi-drug resistance (MDR), which is a resistance to a broad spectrum of structurally unrelated cytotoxic drugs. MDR is mediated by membranal xe2x80x9cpumpsxe2x80x9d which actively expel chemotherapeutic drugs from the tumor cells. Two drug pumps commonly found in cancer are P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP). MDR significantly limits the efficacy of many cancer chemotherapy regimens-and is a major factor in the failure of cancer chemotherapy.
It would, therefore, be most advantageous to have drug derivatives that are targeted or selectively active in the diseased cells rather than in the healthy cells, thus reducing undesirable side effects. It would also be desirable to generate new anti-proliferative agents that overcome drug-resistance, as well as agents that are active as cytotoxic drugs but lack or have a reduced ability to provoke MDR phenotype.
The use of prodrugs to impart desired characteristics such as increased bioavailability or increased site-specificity for known drugs is a recognized concept in the state of the art of pharmaceutical development. The use of various lipids in the preparation of particular types of prodrugs is also known in the background art In none of those instances are the prodrugs able to achieve preferential accumulation of an anti-proliferative drug within the diseased cells of the organ, by activation with intracellular lipases. Rather, the prodrugs enable the drug to be transported to a specific site, or to be released within a specific organ, as in the case of the phospholipid pro drugs of salicylates and non-steroidal anti-inflammatory drugs disclosed in International Patent Application WO 91/16920 which, taken orally, protect the gastric mucosa and release the active principle in the gut.
In other examples of phospholipid prodrugs, the formulation of the prodrugs into liposomes or other micellar structures is the feature that enables their preferential uptake, for instance by liver cells or by macrophages as in the case of the phospholipid conjugates of antiviral drugs disclosed in International patent applications WO 93/00910 and WO 90/00555.
U.S. Pat. No. 5,411,947 discloses a method of converting a drug to an orally available form by covalently bonding a lipid to the drug. It discloses a lipid prodrug of 5-fluorouridine, but differs from the present invention in that the drug is attached to the lipid at the R3 position.
U.S. Pat. No. 4,772,594 discloses prodrugs containing 5-Fluorouracil, but differs from the present invention in that the drug is attached to cholesterol.
U.S. Pat. Nos. 5,149,794 and 5,543,389 disclose covalent polar lipid-drug conjugates to facilitate the entry of drugs into cells at pharmokinetically useful levels. In contrast to the present invention, the disclosed prodrugs are directed to certain intracellular structures and organelles due to the existence of the polar lipid carrier, and drug release from the lipid conjugate is not a requirement for the drug targeting. Moreover, the lipid-drug conjugates may or may not include a spacer and it is explicitly stated that the conjugates may be pharmacologically active themselves. Though phospholipids included in a list of potentially useful polar lipids, the drug-phospholipid conjugates were clearly never reduced to practice and have not been suggested to be inactive derivatives of the drug.
Mono- and di-esters of methotrexate having short alkyl chains ranging from one to four carbons in length, and MTX mono-esters having alkyl chains of up to sixteen carbon atoms, and their cytotoxic effects on cultured cells in vitro have been disclosed by Rosowsky et. (J. Med. Chem., 1978, 21: 380-386; J. Med. Chem., 1984, 27: 605-609). However, none of the disclosed compounds is a phospholipid derivative of MTX as in the present invention.
Non-lipid analogues of methotrexate have been previously disclosed (for example by Antonjuk, D. et al., 1984 J. Chem. Soc. Perkin Trans. 1 (9) 1989-2003), however the derivatives are monoamides and not phospholipids as in the present invention.
International Application WO 94/22483 describes prodrugs which selectively release pharmacologically active compounds in hyperactivated cells, but does not disclose methotrexate or 5-fluorodeoxyuridine derivatives.
The object of the present invention is to provide agents with improved anti-proliferative properties. It is a further object of the present invention to provide prodrugs of anti-proliferative drugs that undergo preferential activation within the disease-affected cells and tissues.
The prodrugs in accordance with the present invention comprise a phospholipid moiety covalently linked, via a bridging group, to an anti-proliferative drug residue, such that the active species is preferentially released, preferably by enzymatic cleavage, at the required site of action.
Thus, the present invention provides, in a first aspect, a prodrug of the general formula I: 
or a pharmaceutically acceptable salt thereof, wherein:
R1 is a saturated or unsaturated, straight-chain or branched, substituted or unsubstituted hydrocarbon chain having from 2 to 30 carbon atoms;
R2 is H or a phospholipid head group;
Z is saturated or unsaturated, straight-chain or branched, substituted or unsubstituted hydrocarbon chain having from 2 to 15 carbon atoms, which may include cyclic elements, and optionally is interrupted by one or more atoms selected from oxygen and sulfur atoms;
X is a direct covalent bond or selected from the group consisting of O, S, NH and C(O) groups; and
D is a residue of an anti-proliferative drug,
wherein the bound anti-proliferative drug residue is an inactive form of the drug which is selectively activated in cells and tissues with elevated phospholipase activity.
According to a currently preferred embodiment, R1 of the prodrug of the general formula I is a hydrocarbon chain having from 5 to 20 carbon atoms, more preferably 15 or 17 carbon atoms.
In another preferred embodiment R2 of the prodrug of the general formula I is selected from the group consisting of choline, ethanolamine, inositol and serine. Preferred anti-proliferative drugs used in the prodrug of the general formula I are methotrexate and 2xe2x80x2-deoxy-5-fluorouridine.
Currently more preferred prodrugs according to the invention are selected from the group consisting of:
1-Stearoyl-2-[3-(xcex1-MTX amido)-Propanoyl]-sn-Glycero-3-phosphocholine,
1-Stearoyl-2-[3-(xcex3-dodecylate-xcex1-MTX amido)-Propanoyl]-sn-Glycero-3-phosphocholine,
1-Stearoyl-2-[4-(xcex1-MTX amido)-Butanoyl]-sn-Glycero-3-phosphocholine,
1-Stearoyl-2-[6-(xcex1-MTX amido)-Hexanoyl]-sn-Glycero-3- phosphocholine,
1-Stearoyl-2-[8-(xcex1-MTX amido)-Octanoyl]-sn-Glycero-3- phosphocholine,
1-Stearoyl-2-[8-(xcex3-dodecylate-xcex1-MTX amido)-Octanoyl]-sn-Glycero-3-phosphocholine,
1-Stearoyl-2-[3-(xcex1-dodecylate-xcex3-MTX amido)-Propanoyl]-sn-Glycero-3-phosphocholine, and
1-stearoyl-2-[5xe2x80x2xe2x80x3-(2xe2x80x3-deoxy-5xe2x80x2-fluorouridine-5xe2x80x3-)-3xe2x80x2xe2x80x3,3xe2x80x2xe2x80x3-dimethyl]glutaroyl-1xe2x80x2xe2x80x3-sn-glycero-3-phosphocholine.
Currently most preferred prodrugs are:
1-Stearoyl-2-[3-((xcex1-MTX amido)-Propanoyl]-sn-Glycero-3-phosphocholine, and 1-Stearoyl-2-[3-(xcex1-dodecylate-xcex3-MTX amido)-Propanoyl]-sn-Glycero-3-phosphocholine.
In another aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as an active ingredient, a prodrug of the general formula I as defined above. The pharmaceutical composition, in accordance with the invention, may further include one or more additional anti-neoplastic agents.
in a further aspect, the present invention provides the use of a prodrug of the aforementioned formula I, or a pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition.
In still a further aspect, the invention provides methods for treatment of diseases or disorders related to an inflammatory condition such as granulomatous diseases, arthritis, rheumatoid arthritis, multiple sclerosis, systemic sclerosis, asthma, psoriasis, systemic lupus erythematosus, inflammatory bowel syndromes and migraines. Also provided are methods for treatment of diseases and disorders related to a degenerative or atrophic condition, in particular central or peripheral neurological diseases or disorders. The diseases and disorders related to a degenerative or atrophic condition may be selected from, but are not limited to, the group consisting of autoimmune, cerebrovascular and neurodegenerative diseases and disorders such as idiopathic dementia, vascular dementia, multi-infarct dementia, traumatic dementia, Alzheimer""s disease, Pick""s disease, Huntington""s disease, dementia paralitica, Parkinson""s disease, diabetic neuropathy, amyotrophic lateral sclerosis, ischemia of the optic nerve, age-related macular degeneration, stroke and trauma. Also provided are methods for treatment of diseases or disorders related to uncontrolled cell growth. Said diseases or disorders may be selected from, but are not limited to, psoriasis, lymphocytic leukemia, myelogenous leukemia, Burkitt""s lymphoma, non-Hodgkin""s lymphomas, mycosis fungoides, osteosarcoma, hydatidiform mole, trophoblastic diseases such as chorioadenoma destruens and choriocarcinoma, and carcinomas of the head and neck, breast, liver, lung, colon, ovary, cervix, urinary, bladder, prostate, pancreas, skin, the gastrointestinal tract and the oropharyngeal areas.
The aforementioned methods comprise administering to a patient in need thereof a pharmaceutical composition containing a therapeutically effective amount of a prodrug of the general formula I, in accordance with the invention.
In a preferred embodiment, the prodrugs of the invention are useful for the treatment of neoplastic growths. The neoplastic growths may be primary or secondary tumors, including drug-resistant tumor, for example tumors resistant to methotrexate and multidrug-resistant tumors.